The in vivo effectiveness of the combination of imiquimod and curcumin nanoemulgel was compared to the imiquimod nanoemulgel and imiquimod gel formulation through topical application for ten days in BALB/c mice. The combination of curcumin with imiquimod in the nanoemulgel system prevented the appearance of psoriasis-like symptoms compared with the imiquimod nanoemulgel and imiquimod gel formulation entirely. Further, the imiquimod nanoemulgel as a mono-preparation slowed and reduced the psoriasis-like skin reaction when compared with the conventional imiquimod gel, and that was contributed to by the control release property of the nano-encapsulation approach.The current investigation aimed to improve the topical efficacy of imiquimod in combination with curcumin using the nanoemulsion-based delivery system through a combinatorial approach. Co-delivery of curcumin acts as an adjuvant therapeutic and to minimize the adverse skin reactions that are frequently associated with the topical therapy of imiquimod for the treatment of cutaneous infections and basal cell carcinomas. The low-energy emulsification method was used for the nano-encapsulation of imiquimod and curcumin in the nanodroplet oil phase, which was stabilized using Tween 20 in an aqueous dispersion system. The weak base property of imiquimod helped to increase its solubility in oleic acid compared with ethyl oleate, which indicates that fatty acids should be preferred as the oil phase for the design of imiquimod-loaded topical nanoemulsion compared with fatty acid esters. The phase diagram method was used to optimize the percentage composition of the nanoemulsion formulation. The mean droplet size of the optimized nanoemulsion was 76.93 nm, with a polydispersity index (PdI) value of 0.121 and zeta potential value of −20.5 mV. The optimized imiquimod-loaded nanoemulsion was uniformly dispersed in carbopol 934 hydrogel to develop into a nanoemulgel delivery system. The imiquimod nanoemulgel exhibited significant improvement ( Imiquimod (IMQ) is a topical immunomodulator that aids in the treatment of superficial basal cell carcinoma, genital warts, and actinic keratosis ,2. IndeeCurcumin (CUR) is a polyphenolic compound of natural origin obtained from the rhizome of Curcuma longa. It is widely reported for various therapeutic activities such as anti-inflammatory and anti-cancer activity . CUR modCurrently, nanotechnology-mediated drug delivery is a leading approach to achieve controlled drug release, co-delivery of primary and adjuvant therapeutics, protecting/minimizing the drug-associated adverse events, and improving the overall drug biopharmaceutical performance ,10. For Nanoemulsion (NE) as a drug delivery vehicle is a widely explored method to enhance the biopharmaceutical performance of poorly soluble therapeutics for topical application. It is easy to process and manufacture, with competitive stability thus generating further interest in the development of NE-based topical formulations. NE consists of colloidal oil droplets, ranging in size between 20 and 200 nm, dispersed in an immiscible aqueous medium . Due to The encapsulation of IMQ within an NE system will allow a deeper skin permeation and protects the skin from the direct contact of IMQ. Moreover, the incorporation of IMQ-loaded NE into hydrogel systems (as a secondary vehicle) will result in the precise control of IMQ released from the NE system and minimizes the IMQ-associated skin reactions . This work aims to improve the topical delivery of IMQ and to reduce the associated adverse effect. The IMQ-NE formulation was optimized to improve the topical delivery of IMQ. Further, CUR-NE was added to the formulation in order to reduce the IMQ-associated skin reaction side effect. Both formulations were incorporated in a hydrogel system to formulate a nanoemulgel (NEG) . Tween 80 (polyoxyethylene sorbitan monooleate), Tween 20 (polyoxyethylene sorbitan monolaurate), PG (propylene glycol), and PEG (polyethylene glycol) 400 were purchased from Merck . Water was obtained from a Milli-Q-water purification system . All the other chemicals were of analytical grade. IMQ (>98.0%) was purchased from Tokyo Chemical Industry, Japan. Oleic acid was purchased from Loba Chem . Curcumin (>97.0%), cremophor EL, IPA , and ethanol were purchased from Sigma Aldrich (Germany). Transcutol HP (diethylene glycol monoethyl ether) was purchased from Gattefose (France). CaptexIn specific oils, surfactants, and co-surfactants, the solubility of IMQ was determined by the addition of an excess of the drug to specified components (2 mL) in stopper vials separately and then mixing with a vortex mixer. The samples were equilibrated in an isothermal water shaker (25 ± 0.5 °C) for 48 h . The equThe phase behavior study was carried out by the construction of phase diagrams . For theThe NE formulations selected from the phase diagram study were centrifuged at 3000 rpm for 30 min. Those formulations that did not show any phase separation were taken for a heating and cooling cycle. Samples were subjected to three cycles between the temperatures of 4 and 45 °C for 48 h to induce stress conditions. Then, the stable formulations were subjected to freeze–thaw cycles. Three freeze–thaw cycles between -21 and +25 °C were carried out to induce stress conditions . The theThe sample was diluted with distilled water (1:100) and mixed for one minute before analysis. The %T was determined using a UV-spectrophotometer at a wavelength of 638.2 nm . The viscosity of the selected IMQ-NE was determined using a Bohlin rotational viscometer . The viscosity of the selected samples was calculated without dilution at room temperature.IMQ-NE formulations were diluted in methanol (100 μL of sample dilutes 1000-fold). The IMQ content was quantified using a UV–visible spectrophotometer at 226 nm. The droplet size and the zeta potential of IMQ-NE were determined by the dynamic light scattering (DLS) technique using a Zetasizer . The samples were diluted with distilled water (1:100) and mixed for one minute before analysis. An accurately weighed quantity of CUR was completely dissolved with the help of vortex mixing in a homogeneous phase of oil and Smix. After that, the aqueous phase was added and subjected to vortex mixing for a few minutes to obtain a clear, transparent, stable, isotropic system in the form of NE similar to the preparation of IMQ-NE described in w/v albumin and 5% methyl alcohol as a co-solubilizer to maintain sink conditions overnight to saturate the condition. The condition was controlled under constant stirring at 37 °C. A 5 mL sample was collected at specified time intervals: 0, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h. Each sample was replaced by a fresh medium to maintain the sink conditions. The concentration of IMQ in each sample was determined using a UV–visible spectrophotometer. Furthermore, the release study of the combination of IMQ-NE and CUR-NE (mixed in a 1:1 ratio) was also performed similarly.The in vitro release study of IMQ from the selected IMQ-NE formulation and IMQ aqueous suspension were carried out (n = 3) using a dialysis tube  with a molecular weight cut-off of 12-14 kD . The dia® 934 (0.5% w/v) was dispersed into a small portion of water with uniform homogenization. Then, For IMQ-NEG, IMQ-NE was incorporated into the Carbopol 934 to give a final concentration of 0.5% IMQ in the gel. For IMQ-CUR-NEG, a mixture of IMQ-NE and CUR-NE was added to dispersed Carbopol® 934 in a 1:1 ratio to give a final concentration of 0.5% of IMQ and CUR in the gel. IMQ containing NEG (IMQ-NEG) and IMQ, along with CUR as a combination NEG (IMQ-CUR-NEG), were prepared. First, CarbopolThe formulations were neutralized by adding 2–3 drops of triethanolamine, and the final pH was adjusted to 5.5. Glycerol was added as a plasticizer in the hydrogel. It was kept overnight to remove entrapped air and allow cross-linking of the polymer to convert it into a gel. The IMQ-NEG and IMQ-CUR-NEG were characterized regarding their rheology, spreadability, extrudability, content uniformity, and droplet size distribution as well as zeta potential.The rheological properties of IMQ-NEG were studied using a rotational parallel plate viscometer  at 25 ± 0.5 °C. The shear-stress profile (15-200 to 200-15 Pa in 60 steps with 10 s equilibration time at each point) and the thixotropic behavior of IMQ-NEG were determined . The BohThe spreadability of the developed IMQ-NEG formulation was evaluated according to a reported method with slight modifications . The samTo ensure the homogeneity of IMQ in the developed NEG formulation, an accurately weighed quantity of the formulation (0.5 g) was taken from three different locations of IMQ-NEG. Each portion of the sample was extracted with 1% albumin in methanol (10 mL) for 30 min. The extracted sample was centrifuged (3000 rpm for 15 min) and the supernatant filtered for the content analysis of IMQ using a UV–visible spectrophotometer. The analysis in terms of the average content and drug percentage was carried out in triplicate .The pH of IMQ-NEG was determined using a digital pH meter . For this, IMQ-NEG (2.0 g) was dissolved in 25 mL of double-distilled water. The pH electrode was then dipped into the dissolved NEG solution, and the constant reading was noted in triplicate.Abdominal skin in full-thickness was obtained from male albino rats weight 200–250 g). Skin hair was carefully removed using an electric razor. The subcutaneous tissue was surgically removed, and isopropyl alcohol was used to clean away adhered fat at the dermis side. Then, distilled water was used to clean the skin and visually inspected for integrity and stored in a deep freezer until use 0–250 g. .2, and the capacity of the receiver cell was 10 mL . Th. Th23]. 2) . Overallw/v Carbopol® 934 gel to obtain a 0.5% w/w concentration of IMQ in the gel. IMQ-CUR-NEG also was prepared by dispersing IMQ-NE and CUR-NE in a 1:1 ratio in 0.5% w/v Carbopol® 934 gel to obtain a 0.5% w/w concentration of IMQ and CUR. The effect of IMQ-NE incorporation into the hydrogel system in terms of its mean droplet size and PdI was evaluated. The results showed no significant change in the mean droplet size (78.39 nm) or PdI (0.254) after the incorporation of IMQ-NE into the gel system.IMQ-NEG was prepared by dispersing IMQ-NE in 0.5% 2/g), IMQ-NEG (0.85 ± 0.04 cm2/g), and IMQ-CUR-NEG (0.87 ± 0.02 cm2/g) were found to be equivalent and did not differ    . CUR ski µg/cm2) . The cumThe percutaneous drug flux (Jss) of the IMQ from IMQ-NEG (0.042 ± 0.01) and IMQ-CUR-NEG (0.071 ± 0.01) was about ten-fold the Jss of IMQ from the IMQ-CUR gel (0.004 ± 0.001) . The Jss-3) and IMQ-CUR-NEG (0.0806 x 10-3) was significantly higher than the Kp of IMQ from the IMQ-CUR gel (0.45 x 10-6) (-3) was significantly higher than the Kp of CUR from the IMQ-CUR gel (0.7 x 10-3). Therefore, the permeation enhancement ratios (ER) of IMQ obtained from IMQ-NEG and IMQ-CUR-NEG were 9.54 and 16.19, respectively. Further, the enhancement ratio of CUR obtained from IMQ-CUR-NEG was found to be 4.36. The permeability coefficient (Kp) of the IMQ from IMQ-NEG (0.0047 x 10 x 10-6) . The Kp IMQ is a widely used drug for the treatment of different types of skin cancer and known to induce psoriasis-like symptoms in BALB/c mice ,3. The aPsoriasis-like symptoms like scaling, redness, and skin thickening started to appear on BALB/c mice treated with IMQ gel from the second day and progressively worsened until the tenth day of the experiment . BALB/c The BALB/c mice were sacrificed on day 11. The treated skin area was collected for histopathology. The histopathology results are in parallel with the in vivo results. The application of the IMQ gel induced aggressive psoriasis-like symptoms , which iIMQ is amongst the most commonly used chemotherapeutic agents for skin cancer ,45. FurtThe use of oleic acid (fatty acid) successfully increases the nanoencapsulation of the weakly basic imiquimod to formulate a nanoemulsion system for controlled topical delivery through the skin. The combinatorial approach with CUR was found to be effective in preventing the adverse skin reactions that are frequently associated with the topical use of imiquimod. This result likely occurs due to the encapsulation of imiquimod into the nanoemulgel delivery system, which resulted in controlled drug release when in contact with the skin as well as the co-delivery of curcumin leading to the complete suppression of psoriasis-like symptoms after the topical application.


For every coating, maximum adhesive strength and excellent self-healing character (three times) were found using 6.775 mol% mixed with low-molecular-weight-based polyols (PU-11-3-3). Defined 1.0 wt% CeO2 was also used for the PU-11-3-3 coating  to obtain UV shielding properties. Both the in situ polymerization and blending processes were separately applied during the preparation of the PU-11-3-3-CeO2 coating dispersion. The in situ polymerization-based coating  showed similar self-healing properties. The PU-11-3-3-CeO2-P coating also showed excellent UV shielding in real outdoor exposure conditions.We prepared a series of polyurethane (PU) coatings with defined contents using poly(tetramethylene oxide)glycol (PTMG) with two different molecular weights (i.e.,  An organic coating deposited on a substrate surface can enhance the protection of that surface, which can ultimately increase the lifespan of that substrate and its structures. These coatings can provide long-term performance, giving them priority in industry ,2,3,4. CIt is common for a coating to be scratched or damaged during its service life. In such conditions, the coating loses its protective property rapidly. To repair the scratch or damage, the coating should heal properly. To heal the coating, a common trend is to give the coating a self-healing property. Self-healing can be defined as the property by which a coating can heal from damage without external activity. There are different ways to make a coating self-healable. Both intrinsic and extrinsic processes are used in the coating industry. In the extrinsic process, additional self-healing agents are added during the coating dispersion preparation. The self-healing agent is released when there is dry coating damage in a real application. In the intrinsic process, no additional agent is used to confer the healing property. The coating obtains this property from hydrogen bonds, van der Waals forces, and the supramolecular structure ,10,11,12There are several varieties of organic coatings used in outdoor atmospheric conditions. A common trend is to use multiple coatings to protect the structure. These can be classified as a primer, mid-coat, and top coat. Different epoxy compounds are mainly used as a primer and mid-coat ,2,3. PolPolydimethyl siloxane (PDMS) is a popular polyol in PU coatings. It has been widely used to improve coating hydrophobicity, corrosion resistance, and antifouling resistance. However, the performance highly depends on the PDMS molecular weight and its contents. In fact, improved protective properties have been recorded using PDMS and other polyether or polyester polyols in PU ,16,18.2, ZnO, and TiO2 are widely being used as UV shielding materials in PU coatings [2, similar to other metal oxides, is an attractive choice as it is significantly less toxic, cheap, and possesses filler properties [A PU coating can easily be degraded in the presence of UV rays. To slow down UV degradation, mixing a variety of nanoparticles into the coating can improve UV degradation resistance. Different nanoparticles  are widely used to enhance coating resistance against UV degradation. However, the effects of nanoparticle content are quite confusing, and only certain contents can improve this property. Saudi Arabia is geographically located in such conditions, where high temperature and high humidity are common during the summertime. Additionally, marine aerosols create harsh conditions in and around coastal areas. Thus, an early failure of organic coatings is common in Saudi Arabia. Rahman et al. found pristine PU coating degradation within a few months during open atmospheric conditions. They also used functionalized multiwalled carbon nanotubes (MWCNTs) in polyurethane (PU) to improve UV degradation resistance. However, UV degradation did not stop completely and was instead slowed down heavily when using 2.0 wt% carboxyl-functionalized MWCNTs. After 1 year, the PU coating with 2.0 wt% carboxyl-functionalized MWCNTs had slightly degraded. Recently, Rahman et al. showed improved UV degradation resistance for PU coatings by adding different metal oxides. The metal oxides act as a UV shielding, and hence, the UV degradation is reduced. The CeOcoatings ,7,19. Thcoatings ,24,25,26Rahman et al. also explained different methods of adding metal oxides to PU coatings. Many factors affected homogeneous metal oxide mixing in the PU coating. The most widely used methods for homogeneous metal oxide dispersion include metal oxide functionalization, sonication during mixing, and proper metal oxide content. Mainly, blending and in situ polymerization are considered to make homogeneous metal-oxide-based PU dispersions .UV degradation resistance and the self-healing of polymers has also been separately worked on ,29,30,31nM = 2000. The dispersion was used to coat mild steel substrates. All coatings were considered for (1/2/3/4) self-healing. The basic coating properties, such as adhesive strength and hydrophobicity, were also evaluated. The promising coatings (PU-11-3-3) were examined for their UV degradation resistance. To enhance UV degradation resistance, a defined CeO2 content (1.0 wt%) was mixed with PU via either blending or in situ polymerization. These coatings were also considered for a self-healing test to evaluate the effect of CeO2 on self-healing properties. The best-performing self-healed coatings were further considered for their UV shielding in real outdoor atmospheric conditions.In this report, a series of PU dispersions see  was syntnM = 650 and 2000), PDMS (nM = 550), triethylamine, N-methyl-2-pyrrolidone, 4,4-dicyclohexylmethane diisocyanate, ethylene diamine, dimethylolpropionic acid, methylethylketone (MEK), and dibutyltin dilaurate were all received from Sigma Aldrich, St Louis, Mo, USA. CeO2 nanoparticles were prepared in accordance with our previous report [Poly(tetramethylene oxide) glycol (s report .12MDI. The MEK (10.0 wt%) was added into the pre-polymer to reduce viscosity. Later, the pre-polymer was neutralized, dispersed, and chain extended by using the proper amount of TEA, H2O (70.0 wt%), and EDA (mixed with 10 ml water). The pre-polymer formation and reaction termination were monitored via FT-IR analysis.The basic polymerization steps were kept the same during the preparation of all dispersions based on previous reports. In all cases, the pre-polymer was prepared by charging PTMG, DMPA solution with NMP and H2. The CeO2 (1.0 wt%) was premixed with acetone (15 mL) and ultrasonicated for 30 min. The CeO2–acetone mixture was added into a premixed DMPA–NMP solution and stirred further for 30 min at 45 °C. This solution was added into a polyol and then charged with H12MDI.All of the polymerization steps were kept exactly the same, except for the addition of CeO2 (1.0 wt%) was premixed with acetone (15 mL) and ultrasonicated for 30 min. Then, the CeO2–acetone mixture was added directly into a PU dispersion. The mixture was stirred for 1 h at 30 °C.The CeOThe coating on the substrate was fixed at 100 μm under wet conditions. The coating was made using a bar auto-coater. All prepared wet coatings were dried at ambient conditions for 48 h. To remove the used solvents completely, the earlier dried coatings were further dried for 12 h at 65–70 °C in an oven.The self-healing test was conducted at 50 °C. Each coating was cut by a sharp blade. To heal the coating, the tested coating was kept in an oven for a maximum of 12 h. The healing was checked via optical microscopy after every 1 h.Different techniques were applied to identify coating properties. In all cases, the value was the average of five testing results. The reaction monitoring was performed and the final polymer was identified via FT-IR  analysis. The photographs of coatings were obtained with a Hitachi S-4200  field emission scanning electron microscope (FESEM) at 5 kV. A Theta Optical tensiometer instrument  was used to obtain the water contact angle. The ASTM D4541 method was followed to evaluate adhesive strength using the pull-off test. XPS  analysis was done to characterize the surface of the coating. The ASTM D2240-75 specification was followed to evaluate the Shore A hardness test using the LX-A rubber Hardness Tester . An exposure test was used to check the UV shielding property of the coating and was based on our previous report .2 content was also used in PU-11 coatings. The different steps of PU preparation were monitored via FT-IR analysis  and urea (NHCOO), as the peaks appeared at 3400 cm−1 (for NH groups) and 1706 cm−1 (for CO groups). A peak at 816 cm−1 for the CH3 group attached with Si was also recorded for coatings using PDMS and thus confirmed the presence of PDMS in PU. Moreover, for PU–CeO2 coatings, a new peak appeared at 560 cm−1, which also confirmed the presence of CeO2 in the PU coatings. The peak at 560 cm−1 was similar for the coatings prepared via blending or polymerization. It was not possible to identify the interaction between the CeO2 and PU groups clearly, as the PU–CeO2 coatings were prepared via two processes. XPS analysis was used to evaluate the effect of the blending and polymerization preparation techniques on CeO2 and PU interactions  than the other coatings. The fastest healed time was found to be 7 h for the PU-9-9-0 coating. As all coatings showed self-healing characteristics the first time, they were also considered for healing a second time. This time, the PU-18, PU-13, and PU-15 coatings failed to heal during the defined duration. The scratches in the PU-18, PU-13, and PU-15 coatings remained after that defined (12 h) duration. The PU-18, PU-13, and PU-15 coatings were comparatively rigid in nature. The rigid structure opposed chain mobility, and ultimately, the coating did not gain a self-healing nature. Interestingly, the PU-9 and PU-11 coatings showed self-healing characteristics the second time as the scratch disappeared within 12 h. In the PU-9 and PU-11 coatings, the flexibility was sufficient to regain the shape, as the scratch healed within that time. The proper balance of polyols made it easy to switch the polymer chain to repair the damage. The PU-9 and PU-11 coatings were also considered for a third healing test. The typical optical photographs are shown in 2. As mentioned before, blending and in situ polymerization were used to include the CeO2 content , wherein the specimen hugely corroded. The coating color changed to yellowish. Some part of the coating was also delaminated. This implies that without a UV shielding material, the coatings could not be used for UV protection. The other coatings were almost free of discoloration, brittleness, delamination, and corrosion. Typical photos of healed exposed coatings are shown in 2 had good UV shielding capacity. No significant difference was seen for these coatings. All coatings were considered for further XPS analysis. Almost the same peak appeared at the same position, confirming that they were least affected under exposure conditions. A typical deconvulated curve analysis was also done for a specific C1s area. It has been reported before that a peak appears at around 289 eV for the CO group due to chain scission [2 coating showed promising results in self-healing and UV-shielding, the operational temperature for self-healing is quite high. The self-healing time is also longer. Above 1.0 wt% CeO2 content also can affect the self-healing and UV-shielding properties. Currently, our group is working to solve these issues.To evaluate the UV shielding resistance, only five coatings  were considered. Visually, it was not possible to verify the coatings’ resistance against UV degradation in the very early stages. When a coating rapidly degraded, which thus initiated metal corrosion, the degradation impact was indicated by color change, pit appearance, and delaminating. The PU-11-3-3 coating was without CeOscission . As the 2 content, as well as an in situ polymerization application. The UV degradation resistance was improved by the addition of a small CeO2 content (1.0 wt%) without sacrificing the basic protective properties of hydrophobicity and adhesive strength. A small content (6.775 mol%) of mixed low-molecular-weight polyols contributed to 3 times better self-healing. The healed coating also showed excellent UV degradation resistance for open atmospheric conditions. This type of coating can be useful for outdoor applications.A coating with multi-time self-healing and UV-shielding properties  was prepared using a proper combination of polyols and CeO


The aim of the present study is to unveil the role of 5-HT6 receptors in the VLO and the underlying mechanisms in pain modulation. Here, by using the spared nerve injury (SNI) pain model, first, we report that 5-HT6 receptor protein decreased in the contralateral VLO compared with the ipsilateral VLO in rats with allodynia. Second, microinjection of the selective 5-HT6 receptor agonists EMD-386088 and WAY-208466 into the contralateral VLO consistently and significantly depressed allodynia. Third, microinjection of the selective antagonist SB-258585 blocked the agonist-induced anti-allodynic effect, while the antagonist applied alone to the VLO had no effect. Furthermore, the anti-nociceptive effect of EMD-386088 on neuropathic pain was prevented by the adenylate cyclase (AC) inhibitor SQ-22536, and protein kinase A (PKA) inhibitor H89, suggesting that AC/PKA signaling might underlie the antinociception of agonists. Finally, the 5-HT6 receptors were found to be colocalized with a glutamate transporter (EAAC1) by immunofluorescent staining, and the glutamate receptor antagonist kynurenic acid was found to completely block antinociception. These findings indicated that the antinociceptive effect of 5-HT6 receptor agonists might occur via interaction with the glutamatergic system. Altogether, the agonists activated 5-HT6 receptors present in the glutamatergic neurons in the VLO to facilitate the AC/PKA cascade, which subsequently might evoke glutamate release, thus depressing allodynia. These findings suggest a potential therapeutic role of 5-HT6 receptor agonists in treating neuropathic pain.Mechanical allodynia, characterized by a painful sensation induced by innocuous stimuli, is thought to be caused by disruption in pain-related regions. Identification and reversal of this pathologic neuroadaptation are therefore beneficial for clinical treatment. Previous evidence suggests that 5-HT Neuropathic pain is a maladaptive pain condition that has become a public health challenge . This co6 subtype is of special interest. Mounting evidence suggests that both 5-HT6 receptor agonists and antagonists display pro-cognitive properties  fibers from the dorsal raphe nucleus (DR) . Growingoperties , such aseceptors , and 5-H effects . Such pasettings . A pioneagonists . Althoug6 receptors in the VLO in neuropathic pain model and (ii) to examine the mechanistic underpinnings of 5-HT6 receptors affecting nociceptive behaviors. Using this framework, we speculate on whether 5-HT6 receptors represent a platform for the development of therapeutic interventions in neuropathic pain.Therefore, the present study was conducted (i) to examine the role of 5-HTad libitum. All animals were acclimatized for 3 days before any experimentation. The experimental protocols and animal procedures were approved by the Institutional Animal Care Committee of Xi’an Jiaotong University and were carried out in strict accordance with the ethical guidelines of the International Association for the Study of Pain  were used for all experiments and were provided by the Medical Experimental Animal Center of Xi’an Jiaotong University, Shaanxi Province, China. The animals were group housed under a 12-h/12-h light/dark cycle with access to food and water  of Pain  for the The spared nerve injury (SNI) model was implemented as previously described . BrieflyThe rats were anesthetized through i.p. injection of sodium pentobarbital (50 mg/kg). Then the head was immobilized in a stereotaxic frame. A small craniotomy was performed just above the VLO, contralateral to the affected paw, since a majority of the nociceptive signals are transmitted to the contralateral VLO. The coordinates for the unilateral VLO microinjection were as follows: AP + 4.2 mm, ML + 2.0 mm, DV – 4.6 mm . A stainThe paw withdrawal threshold (PWT) was quantified in response to mechanical stimulation (von Frey filaments) using the up-down method . The ani6 receptor agonists EMD-386088 , a very short-acting inhalation anesthetic (approximately 1–2 min), and a 1-μl microsyringe with the tip extending 2 mm beyond the end of the guide cannula was inserted into the VLO through the guide cannula. For double-administered agents, the interval between the two injection was 10 min. Over a 60-s period, 0.5 μl of drug dissolved in saline or 10% dimethyl sulfoxide (DMSO) was slowly infused at constant speed, and after the rats recovered from the enflurane anesthetized, effects on allodynia were observed over a 60-min period following drug infusion. Values from each treated group were obtained from different experiments . The folD-386088  and WAY-D-386088 , selectiB-258585 , non-selB-258585 , adenylaB-258585 , and PKAB-258585 . WAY-208B-258585  and our 6 receptor agonists into the VLO affects the locomotor activity of animals, an open-field test was conducted after administration of the selective 5-HT6 receptor agonist EMD-386088 or the vehicle into the VLO of the SNI rats. Video-tracking software  was used to test individual rats. Testing boxes  were set in an isolated dark room, and two standard laboratory lamps were fixed above the boxes for illumination. The room was kept quiet and dimly lit during the whole experiment. After the rats were gently placed in the boxes, their locomotor activities were recorded by a digital camera (SONY) and analyzed offline by the horizontal trajectories reflecting the animals’ locomotor activity. The videos were recorded using digital video cameras mounted above the testing boxes.To explore whether microinjection of 5-HTg for 15 min at 4°C. The supernatant was collected and stored at −80°C until use. Sample protein levels were measured using a bicinchoninic acid (BCA) protein assay. Loading proteins were separated on acrylamide gels and then transferred to pore size 0.45 μm polyvinylidene difluoride (PVDF) membranes . After blocking with 5% non-fat milk, the membranes were probed for anti-rabbit 5-HT6 receptor , and anti-mouse actin . Densitometry of each protein was evaluated by Image-J (NIH). Samples from each animal were run at least four times to minimize inter-blot variance. Raw values were normalized to actin.Rats went through procedures in After the behavioral experiment, the animals were deeply anesthetized with chloral hydrate (400 mg/kg) and the microinjection sites were marked with Pontamine Sky Blue dye . Rats were transcardially perfused with 0.01 M PBS (pH 7.4), followed by 4% paraformaldehyde solution . The brains were then removed and fixed in 4% PFA for 24 h, then placed in 30% sucrose solution for 3 days at room temperature. Subsequently, the brains were embedded in optimum cutting temperature medium  and cut into 40 μm thick sections using a Leica CM3050 S cryostat. Then the slices were mounted on gelatin-covered slides and stained with cresyl violet. The injection sites were histologically identified to be within the VLO or not. Only the data with explicit corresponding histology representing presence within the VLO were used for final data analysis and 15 rats were excluded from data analysis. To keep consistent, we dissect it from bregma +4.2 to + 3.24 . A schem6 receptor  and anti-goat neuronal glutamate transporter  for 48 h at 4°C. Fluorescent secondary antibodies (Invitrogen/Thermo Fisher Scientific), goat anti-rabbit Alexa 488 and donkey anti-goat CY3 at dilutions of 1:500 and 1:600, respectively, were incubated for 2 h at room temperature. Coverslips were mounted using mounting media with DAPI . Sections were stained at the same time and images of the VLO were taken with the same exposure time. By using Image-J, the intensity threshold was applied to each image to include the positive labeling while minimizing the inclusion of non-specific, background staining in the sections. The experimenters were blind to the experimental conditions during imaging and analysis.For immunofluorescence, rats went through procedures in post hoc test or Student’s t-test was further conducted as needed. To determine differences between two groups, Student’s t-test was performed. P < 0.05 was considered to be statistically significant. All values were expressed as mean ± SEM.All values were expressed as mean ± SEM. One-way ANOVA or two-way ANOVA analyses were performed using IBM SPSS statistics 19.0 . The areas under the time-course curve (AUC) of the PWT during the 60-min observation period were counted. For results with significant interaction effects in two-way ANOVA, a simple effect test was conducted for further analysis. For results without significant interaction effects, Bonferroni’s T-test  showed that the PWT in the sham group (14.14 ± 0.4773) was not different from that in the naïve group (14.98 ± 0.1509). Therefore, the sham was used as the control in the following experiments  = 9.156, p = 0.0007], and post hoc analyses showed a significantly lower protein level of 5-HT6 receptors in the VLO contralateral to the affected paw (0.729 ± 0.067), compared to those of the ipsilateral VLO  and control  groups  groups . These r6 receptor expression in the contralateral VLO, we first microinjected selective 5-HT6 receptor agonist EMD-386088  into the contralateral VLO. We found that EMD-386088 (5 and 10 μg) reduced SNI-induced allodynia as the PWT was increased  = 13.40, p < 0.0001], across times  and for treatment × time interaction . The detailed comparisons at individual time points between various groups are also shown in p < 0.01 and p < 0.0001, respectively). In addition, the effects of 10 μg EMD-386088 were significantly larger than the 2.5 μg group (p < 0.01). No significant difference was observed between the 5 and 10 μg EMD-386088 groups, as shown in 6 receptor agonist EMD-386088 in the VLO on PWT  or the vehicle (4358.614 ± 533.707) into the contralateral VLO was not significantly different between the two groups during the 60-min observation period  reduced SNI-induced allodynia as the PWT was increased  = 37.90, p < 0.0001], across times  and for treatment × time interaction . Detailed comparisons at individual time points between the various groups are shown in p < 0.05, p < 0.01, and p < 0.0001, respectively). No significant difference was observed between these groups, as shown in 6 receptor agonists on allodynia was due to the activation of 5-HT6 receptors.However, a previous study also reported a weaker effect of EMD-386088 on 5-HTreuptake . To furtncreased . As show6 receptor antagonist SB-258585 (4 μg) into the VLO, 10 min prior to EMD-386088 (10 μg) injection, completely antagonized the EMD-evoked inhibition of allodynia as the PWT was reduced to the control level, as shown in F = 69.73, p < 0.0001], across times  and for treatment × time interaction . Detailed comparisons at individual time points between various groups were shown in p < 0.0001), and no significant difference was found between the SB + EMD and vehicle + vehicle groups, as shown in Microinjection of the selective 5-HTF = 38.08, p < 0.0001], across times  and for treatment × time interaction . Detailed comparisons at individual time points between the various groups are shown in p < 0.01), and no significant difference was found between SB + WAY and vehicle + saline groups, as shown in In addition, microinjection of SB-258585 (4 μg) into the VLO, 10 min prior to WAY-208466 (8 μg) injection, completely antagonized the WAY-evoked inhibition of allodynia, as the PWT was reduced to the control level, as shown in p > 0.05, Microinjection of SB-258585  into the VLO had no effect on allodynia induced by SNI, as the PWT was maintained at the control level  = 27.08, p < 0.0001], across times , and for treatment × time interaction . The detailed comparisons at individual time points are shown in p < 0.0001), while the PWT was reduced to the control level , across times , and for treatment × time interaction . The detailed comparisons at individual time points are shown in p < 0.0001), but no significant difference was found between H89 + EMD, and vehicle + EMD groups (p > 0.05), as shown in Furthermore, microinjection of the PKA inhibitor H89 (10 nmol) into the VLO did not influence allodynia induced by SNI. However, when the same dose of H89 with EMD-386088 (10 μg) was applied into the VLO, the H89 significantly blocked the EMD-evoked inhibition of allodynia . As show6 receptor agonists’ anti-allodynic effect. Considering that the VLO is an indispensable part of the analgesic system, with activation of the VLO attenuating allodynia, and that activation of 5-HT6 receptors inhibited allodynia, we speculated that 5-HT6 receptors might interact with excitatory neurons. To test this hypothesis, we first conducted immunostaining of 5-HT6 receptors and glutamate transporter (EAAC1), as previous immunoreactivity of EAAC1 corresponds to what has been described for glutamatergic neuronal systems  = 25.81, p < 0.0001], across times , and treatment × time interaction . Detailed comparisons at individual time points are shown in p < 0.0001), where the PWT was reduced to the control level [p > 0.05, 6 receptor agonists’ anti-allodynic effect was dependent on glutamate release.To confirm the hypothesis that 5-HT1A receptor and D2-like dopamine receptor via the increase of endogenous opioid peptide, 5-HT and dopamine release presynaptically inhibited the tonic inhibitory action of GABAergic terminals on the neurons projecting to VLO, such a disinhibitory effect leads to activation of the Sm–VLO–PAG brainstem descending inhibitory system and produces antinociception. The present study aimed to dissect the individual effect of 5-HT6 receptors and their interactive substrate within the VLO. Our previous study showed that 5-HT microinjection into the VLO depressed SNI-induced allodynia and that this anti-allodynic effect was significantly antagonized by the selective 5-HT6 receptor antagonist SB-258585  and non-conventional signaling  into the VLO. It is well documented that the 5-HT6 receptors are a type of excitatory Gs protein coupled receptor, and that activation of this receptor type increases neuronal activity , some of which are located on glutamatergic neurons, facilitated glutamate release, thereby indirectly exciting the VLO output neurons projecting to the PAG to induce anti-nociception  assessed mechanical allodynia-like behavior of persistent neuropathic pain  contribution to different types of pain.Notably, several studies have indicated that local peripheral and central 5-HTstration . Several the rat . The stihic pain . As 5-HT6 receptors in the VLO regulated allodynia in a rodent model of neuropathic pain via glutamatergic neurotransmission, as well clarifying the AC/PKA signal pathway. Based on these findings, the present study uncovered a potential therapeutic role of 5-HT6 receptor agonists in the context of neuropathic pain.In summary, the present study reported that activation of 5-HT6 receptor signaling in neuropathic pain, current methodological limitations made it difficult to parse out the contribution of certain neuronal subpopulations in refining cortical sensory processing. Besides, in the present study the 5-HT6 agonist was microinjected into the VLO rather than systemically administered. From clinic perspective, it should be acknowledged that systematic administration might risk potential off-target and contradictory or even unwanted effects, which should be addressed in the future therapeutic development.Although we have provided strong evidence for the involvement of 5-HTThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Institutional Animal Care Committee of Xi’an Jiaotong University.YZ, FH, and CY designed the research. YZ performed the experiments. XY, YWu, JL, and YWa provided technical support and performed the surgery. YZ and JY analyzed the data. YZ and CY wrote the manuscript and FH edited it. All authors approved the manuscript for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Papillary renal cell carcinoma (pRCC) accounting for near 20% of renal cell carcinoma is the second most common histological subtype. MiRNAs have been demonstrated to played significant roles on predicting prognosis of patients with tumors. An appropriate and comprehensive miRNAs analysis based on a great deal of pRCC samples from The Cancer Genome Atlas (TCGA) will provide perspective in this field.We integrated the expression of mRNAs, miRNAs and the relevant clinical data of 321 pRCC patients recorded in the TCGA database. The survival-related differential expressed miRNAs (sDEmiRs) were estimated by COX regression analysis. The high-risk group and the low-risk group were separated by the median risk score of the risk score model (RSM) based on three screened sDEmiRs. The target genes, underlying molecular mechanisms of these sDEmiRs were explored by computational biology. The expression levels of the three sDEmiRs and their correlations with clinicopathological parameters were further validated by qPCR.P < 0.001), eighteen differential expressed miRNAs (DEmiRs) were remarkably related with the overall survival (OS) of pRCC patients. Three sDEmiRs with the most significant prognostic values  were employed to establish the RSM which was certified as an independent prognosis factor and closely correlated with OS. In the verification of clinical samples, the overexpression of miR-410-3p and miR-6720-3p were detected to be associated with the advanced T-stages, while miR-34a-5p showed the reversed results.Based on univariate COX analysis (The study developed a RSM based on the identified sDEmiRs with significant prognosis prediction values for pRCC patients. The results pave the avenue for establishing and optimizing a reliable and referable risk assessing model and provide novel insight into the researches of biomarkers and clinical treatment strategies. With the increasing morbidity, papillary renal cell carcinoma (pRCC) accounting for near 20% of renal cell carcinoma (RCC), has transcended into the second most common histological subtype , 2. FollAlthough Food and Drug Administration (FDA) has approved certain drugs of immunotherapy and targeted therapy, and some of which gained encouraging outcomes for only a few subgroups of patients with pRCC, a wider range of pRCC patients remain in desperate need of the more ideal and promising treatment strategies , 9. FromConsisting of abundant immune molecules and other regulating cytokines, tumor microenvironment (TME) closely associating with the tumor immune response processes, is the crucial concern of discovering biological markers , 11. WitAs a kind of small endogenously expressed RNA molecules with approximately 17–23 nucleotides in length, miRNAs play indispensable roles on the genes posttranscriptional regulations including messenger RNAs (mRNAs) cleavage and protein translation , 16. FurTherefore, we designed the study to discover and validate the roles of miRNAs on clinical prognosis prediction of pRCC through establishing a more personalized and accuracy risk predicting model. Furthermore, the clinical relevance of the miRNAs and the interactions of their target genes were also detected. The ultimate aim of the study is to offer the guiding light for clinical decision.28 pRCC tissues (type 1 and type 2) and adjacent tissues were acquired from the patients who accepted operation in The Affiliated Hospital of Southwest Medical University from September 2018 to December 2019. The acquired samples were frozen in liquid nitrogen immediately and then stored at − 80 °C until miRNAs extraction.https://portal.gdc.cancer.gov/), which contained data from 32 non-tumor tissues and 389 pRCC samples. These data were updated on May 7, 2020. Clinical data about these patients were downloaded and extracted (the OS of patients ≤ 30 days were excluded because of these patients probably died of unpredictable factors). RNA-seq and miRNAs results were combined into matrix files by the use of a merge script in the Perl language (http://www.perl.org/). The R software limma package (https://bioconductor.org/packages/release/bioc/html/limma.html) was used to screen for differentially expressed genes in tumor and adjacent non-tumor tissues. We present all differential mRNAs and miRNAs analysis data with the screening value of “FDR < 0.05, log2| FC | > 1 and P < 0.05”. The differential mRNAs and miRNAs were prepared to the subsequent study.A series of transcriptome RNA-sequencing and miRNA data of pRCC samples including type 1 and type 2 were downloaded from the TCGA data portal (P < 0.05). Hazard ratio (HR) was utilized to specified sDEmiRs into protective and deleterious parts. These sDEmiRs were identified for the subsequent study.Differentially expressed miRNAs (DEmiRs) correlated with survival in patients with pRCC were verified as survival related DEmiRs (sDEmiRs). Univariate COX analysis was employed to screen sDEmiRs ] + [Expression levels of miR-410-3p * (0.4567728)] + [Expression levels of miR-6720-3p * (0.1965725)]. The values of RSM were utilized to evaluate multiplied subtypes of pRCC patients.To further explore the significances of the sDEmiRs, we analyzed the correlation of the RSM and clinicopathologic features, of which the “TNM staging method” is regarded as the most common method to evaluate the tumor status. The maximum tumor diameter and tumor invasion extent were used to divide the T-stage, with the larger tumor sizes and more extensive invasion in the more advanced T stages. “N-stage” reflected the lymph node metastasis status, with more metastatic lymph nodes in the more advanced N stages. “M-stage” was distinguished based on the distant metastasis conditions, and the advanced M stages probably represented the poor tumor outcomes. In addition, “stage” was a comprehensive way combining with T-stage, N-stage and M-stage to separate pRCC patients from I, II, III and IV stage.−ΔCt method. Three replicate assays were performed per cDNA sample.Total RNA was extracted from pRCC tumor and adjacent tissues by TRIzol (Invitrogen). RNA was reverse-transcribed to cDNA by the PrimeScript RT reagent kit . The reaction steps were as follows: 37 °C for 15 min and 85 °C for 5 s. The quantitative polymerase chain reaction (qPCR) was performed on an ABI 7500 Real-Time PCR System (Applied Biosystems) utilizing a SYBR Green assay (TaKaRa). The reaction cycling conditions  were carried out; primer sequences are shown in Table http://www.targetscan.org/vert_72/), miRTarBase (http://mirtarbase.mbc.nctu.edu.tw/php/index.php) and miRDB (http://mirdb.org/). And the filter standard for a target gene was no less than two databases supported it. In order to explore the interaction between these target genes, a PPI network based on the data was acquired on the STRING online database (https://string-db.org/). PPI networks were employed to show the relationships between these target genes. The standard for a core gene was no less than five node degrees. Cytoscape software version 3.7.2 was used to show PPI results. Functional enrichment analysis was performed through the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways to explore the underlying molecular mechanisms of differential IRGs. GO and KEGG pathways were on the basis of R software packages of cluster profiler, org.Hs.e.g.db, and enrichplot.The target genes were selected by the databases of TargetScan , and the ordinate represents the sensitivity (true positive rate). Univariate Cox regression analysis, Pearson correlation analysis and multivariate regression analysis were utilized to confirm the sDEmiRs. Kaplan–Meier curve was employed to estimate the OS of the high-risk group and the low-risk group of pRCC patients. All statistical analysis was conducted by SPSS21.0 software  and GraphPad Prism5 . Variations in clinical parameters were determined via independent 2∣FC∣ and the heatmap was illustrated in Fig. 1244 differentially expressed pRCC genes were screened by limma algorithm, of which 462 were down-regulated and 782 were up-regulated , such as miR-323a-3p, miR-409-5p, miR-34a-5p, miR-539-5p, miR-376c-3p, miR-379-5p, miR-337-3p, miR-410-3p, miR-216a-5p, miR-495-3p, miR-381-3p, miR-382-5p, miR-493-3p, miR-411-3p, miR-519a-5p, miR-6720-3p, miR-105-5p and miR-224-5p. And the relationships between these sDEmiRs and prognosis were illustrated in the forest map, in which miR-34a-5p showed a reverse trend to that of other miRNAs, with the negative correlation with the poor clinical outcomes , of which three sDEmiRs were further selected to establish the RSM which could be served as an independent prognostic factor to evaluate the prognosis of patients with pRCC. In the RSM, the younger patients, and patients with advanced stage, advanced T-stage, advanced N-stage and advanced M-stage may get the higher risk scores. In the analysis of the regulatory networks of target genes of the three sDEmiRs, we found the lower expression levels of SLC34A2, SPATA18, TPK1, CHL1, LRRK2, PHIHIPL and SCEL were related with the longer OS, while the lower expression of TUSC3, TMEM164 and CEBPB were correlated with the poor prognosis. Besides, during the PPI analysis, nine target genes were regarded as the core genes.To further improve the reliability and persuasion for providing clinical decisions, we recruited a considerable number of patients with pRCC. Furthermore, certain specific sDEmiRs  with obvious clinical significances have also been validated to be implicated in the prognosis of pRCC and potentially serving as the molecular bioindicators for forecast and assessment of OS. The further exploration of target genes of these sDEmiRs provided a novel insight for future researches.Although the potency of sDEmiRs for predicting prognosis has been ascertained, and we also validated the expression levels of miR-410-3p, miR-6720-3p and miR-34a-5p in tumor tissues with different T-stages, some defects are still needed to be pointed out and further discussed. Firstly, the absence of the combination with the proteomics and metabolomics decreased the integrity. Additionally, the practical applying values of the RSM based on the three sDEmiRs have yet be adequately elucidated and on desperate need of wide corroboration. Thirdly, except for miR-410-3p, miR-6720-3p and miR-34a-5p, other potential sDEmiRs and their target genes as well as the correlated underlying mechanisms remain to be further explored. Fourthly, we didn’t distinguish the type 1 or 2 papillary RCC. Fifthly, we didn’t split the data into training and testing set because of limited sample size.In the present study, we comprehensively analyzed and validated the effects of sDEmiRs on predicting prognosis of pRCC. The results pave the avenue for establishing and optimizing a reliable and referable risk assessing model and provide novel insight into the researches of biomarkers and clinical treatment strategies.Additional file 1: Table S1. The results of multivariate Cox regression coefficients.Additional file 2: Figure S1. The relationship between the hsa-miR-34a-5p and age. The younger patients were correlated with the lower expression levels of miR-34a-5p.Additional file 3: Figure S2. Survival curve of target genes. Kaplan‐Meier survival curve of target genes. The higher expression of CEBPB (A), TMEM164 (B) and TUSC3 (C) were correlated with the poor prognosis. The higher expression of CHL1 (D), LRRK2 (E), PHYHIPL (F), SCEL (G), SLC34A2 (H), SPATA18 (I) and TPK1 (J) were associated with the longer OS.


Epilepsy is one of the most common and severe brain diseases. The exact cause of epilepsy is unclear. Epilepsy often occurs following brain damage, such as traumatic brain injury (TBI) and ischemia. Cerebrolysin is a porcine brain peptide that is a unique neurotropic and neuroprotective agent. Cerebrolysin has been reported to increase neuroprotective effects after TBI, ischemia, and other CNS diseases. However, the effects of cerebrolysin on seizures are not known. Therefore, this study aimed to investigate the effects of neuropeptide cerebrolysin on neuronal death in the hippocampus after a seizure. To confirm the effects of cerebrolysin, we used a pilocarpine-induced seizure animal model. Cerebrolysin  was immediately injected after a seizure induction. After 1 week, we obtained brain tissues and performed staining to histologically evaluate the potentially protective effects of cerebrolysin on seizure-induced neuronal death in the hippocampus. We found that cerebrolysin decreased hippocampal neuronal death after a seizure. In addition, an increase in brain-derived neurotrophic factor (BDNF) was confirmed through Western blot analysis to further support our hypothesis. Therefore, the present study suggests that the administration of cerebrolysin can be a useful therapeutic tool for preventing neuronal death after a seizure. Epilepsy is one of the most common brain diseases, affecting about 70 million people worldwide . Tempora2. Animal experiments were performed according to the criteria of the Committee on Animal Habitation . We made every effort to minimize the pain of the animals, which were ultimately sacrificed by isoflurane anesthesia.in Vivo Experiments) guidelines.This experiment used Sprague–Dawley male rats  aged 8 weeks. The animal rooms were kept at a constant humidity (55 ± 5%) and room temperature (22 ± 2°C). The room’s lighting was set to automatically switch on at 12 h intervals (on at 6:00 and off at 18:00). This guideline was designed based on the ARRIVE  19 h before the administration of pilocarpine. Scopolamine  was administered 30 min before the administration of pilocarpine . Thirty Experimental groups were classified into four groups: sham-vehicle, sham-cerebrolysin, seizure-vehicle, and seizure-cerebrolysin. To evaluate the effect of cerebrolysin on pilocarpine-induced seizures, cerebrolysin groups were injected with cerebrolysin  intraperitoneally daily for 1 week, 2 h after a seizure induction, and vehicle groups were injected with 0.9% saline intraperitoneally in the same way. Also, to evaluate the anticonvulsant effect of cerebrolysin on pilocarpine-induced seizure, cerebrolysin groups were injected with cerebrolysin  10 min before pilocarpine injection. The present study used this cerebrolysin concentration since several works have demonstrated a significant neuroprotective effect after brain insult .Animals were sacrificed at 1 week after a seizure. Animals were injected with urethane  as anesthesia. After completely entering an anesthetic state, the animals were perfused with 0.9% saline and then 4% paraformaldehyde. The brains were harvested quickly and accurately and were fixed with 4% paraformaldehyde for 1 h. After fixation, the brains were immersed in a 30% sucrose solution as a cryoprotectant for 2 days . Two dayMicroscopy images were obtained with an Olympus IX70 microscope  equipped with a U-HGLGPS  and an INFINITY3 digital camera . We obtained the image using the INFINITY ANALYZE software.2).Live neurons were evaluated by staining for neuronal nuclei (NeuN) to confirm the effect of cerebrolysin on pilocarpine-induced seizure. Following the brain cryostat section, we stained the cut tissue. After precleaning to eliminate the remaining blood cells in the tissues, we put the tissues in monoclonal mouse anti-NeuN antiserum  and kept them overnight for 16 h at 4°C. Sixteen hours later, the tissues were placed in anti-mouse IgG  for 2 h at room temperature and then placed in an ABC complex solution  for 2 h at room temperature. Then, the samples were transferred to slides after 3,3′-diaminobenzidine  coloring for 1.5 min. Slides were then dried and mounted using Canada balsam. The tissues were observed through an Axioscope microscope. Live neurons were averaged by blind quantification. Live neurons were quantified in the stratum pyramidale (SP) of hippocampal cornu ammonis1 (CA1) and CA3 and expressed as the density (cell count/mm2).Microglial cells were evaluated by ionized calcium-binding adaptor molecule 1 (Iba1) to confirm the effect of cerebrolysin on pilocarpine-induced seizure. Following brain cryostat sectioning, we stained the cut tissue. After precleaning to eliminate the remaining blood cells in the tissues, we put the tissues in monoclonal goat anti-Iba1 antiserum  and kept them overnight for 16 h at 4°C. Sixteen hours later, the tissues were placed in Alexa Fluor 594-conjugated donkey anti-goat IgG secondary antibody  for 2 h at room temperature. Then, they were put on the slides. The slides were dried and mounted with DPX , and the tissues were observed through an Axioscope microscope. Microglial cells were quantified in the stratum oriens (SO), stratum pyramidale (SP), and stratum radiatum (SR) of the hippocampal CA1 and CA3. Microglial cells were expressed as their density (cell count/mm2).Astroglial cells were evaluated by the glial fibrillary acidic protein (GFAP) to confirm the effect of cerebrolysin on pilocarpine-induced seizure. Following brain cryostat sectioning, we stained the cut tissue. After precleaning to eliminate the remaining blood cells in the tissues, we put the tissues in monoclonal rabbit anti-GFAP antiserum  and kept them overnight for 16 h at 4°C. Sixteen hours later, the tissues were placed in Alexa Fluor 488-conjugated donkey anti-rabbit IgG secondary antibody  for 2 h at room temperature. Then, the samples were placed on the slides. The slides were dried and mounted with DPX , and the tissues were observed through an Axioscope microscope. Astroglial cells were then quantified in the stratum oriens (SO), stratum pyramidale (SP), and stratum radiatum (SR) of hippocampal CA1 and CA3. Astroglial cells were expressed as their density  and kept them overnight for 16 h at 4°C. Sixteen hours later, the tissues were placed in Alexa Fluor 488 donkey anti-rabbit IgG secondary antibody  for 2 h at room temperature. Then, the samples were placed on slides. The slides were dried and mounted with DPX . The tissues were then observed through an Axioscope microscope. Apoptotic cells were counted by blind quantification in the hippocampal CA1 and CA3 regions.g for 20 min at 4°C, and the supernatant was harvested. The harvested supernatant was incubated at 100°C for 10 min and stored at −80°C in an ultra-low freezer until use. The protein composition within the hippocampus was measured by a Bradford protein assay. Hippocampal proteins were diluted in an SDS electrophoresis sample buffer, separated on a 14% SDS-polyacrylamide gel, and then transferred to a PVDF (polyvinylidene difluoride) membrane. Non-specific binding was prevented by using 5% skim milk and 5% BSA (TNF-α) in TBST  for over 1 h at room temperature. Protein-transferred membranes were incubated on primary antibodies , ABN1381, diluted 1:1,000, Millipore, phospho-CREB (p-CREB), #9198, diluted 1:1,000, Cell signaling, TNF-α, ab6671, diluted 1:500, Abcam) overnight at 4°C in an incubator. After primary antibody incubation, the membranes were washed three times for 5 min in TBST. Afterward, the primary anti-BDNF, anti-TrkB, anti-phospho-TrkB, anti-phospho-CREB, and anti-TNF-α-reacted membranes were incubated for 1 h in anti-rabbit IgG secondary antibody conjugated with horseradish peroxidase . Last, to visualize the protein concentration, we used an ECL (enhanced chemiluminescence) solution  before observation. The ECL solution-mounted membrane was made to react using a chemiluminescence imaging system device . All data were analyzed by Image J.To verify the protein level of BDNF in the vehicle and cerebrolysin groups, we performed a Western blotting analysis. The bilateral hippocampus was obtained and homogenized in a RIPA buffer consisting of 10 mM Tris-HCl (pH 7.4), 1% Non-idet P-40, 150 mM NaCl, 0.5% sodium deoxycholate, and 0.1% SDS. The homogenized hippocampus was centrifuged at 14,000 × post hoc analysis, and the data from the two groups were analyzed by a Mann–Whitney U test. The data are expressed as the standard error of the mean (SEM) and were regarded as significant when the difference was p < 0.05.We used non-parametric tests to determine the statistical significance between the experimental groups. The data of four groups were analyzed by a Kruskal–Wallis test and Bonferroni We confirmed the neuroprotective effects of cerebrolysin treatment on seizure-induced neuronal death. Cerebrolysin was injected for 1 week after pilocarpine-induced seizure . Both grn = 5) for each sham group, and n = 5–7 for each seizure group {∗p < 0.05 vs. vehicle-treated group; #P < 0.05 vs. sham-operated group }.We conducted neuronal nuclei (NeuN) staining to confirm the effects of cerebrolysin on neuronal survival after a seizure. We sacrificed the animals at 1 week after inducing seizure and quantified the density of their live neurons in the hippocampus. As a result, after comparing the density of the live neurons of the seizure-vehicle and seizure-cerebrolysin groups, we found that the density of live neurons was increased in the group treated with cerebrolysin in the hippocampal CA1 and CA3 regions, rather than the seizure-vehicle group . The datn = 5, for each sham group, and n = 5–7 for each seizure group {∗p < 0.05 vs. vehicle-treated group; #p < 0.05 vs. sham-operated group }.Glial activation is increased not only during seizure but also after other diseases . Inflammn = 3–4, for each seizure-experienced group .Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine . To confn = 5–7, for each seizure group {∗p < 0.05 }.Cleaved caspase-3 staining is an immunofluorescent staining method used to confirm apoptosis. We performed cleaved caspase-3 staining to verify the effect of cerebrolysin on apoptosis after a seizure. There was no caspase-3 activation observed in the sham-operated groups. The seizure-experienced group showed an increased level of caspase-3 activation in the hippocampal CA1 and CA3 regions. However, the administration of cerebrolysin showed a decreased number of caspase-3-positive cells in the hippocampal CA1 and CA3 regions compared to the seizure-vehicle group . The datn = 3–4, for each seizure group .BDNF is regarded as a potent neural modulator, which is beneficial to neuronal functions and promotes neuroprotection . To confn = 3–4 for each seizure group .Tyrosine kinase receptor B (TrkB) is known as a receptor for BDNF . To evaln = 3–4 for each seizure group .Phospho-cAMP-response-element-binding (p-CREB) is a transcription factor present downstream of BDNF that promotes neuronal protection and cell survival . We evalIn the present study, we verified that cerebrolysin exerts powerful neuroprotective effects after pilocarpine-induced seizure. Seizure is one of the most common neurological diseases, but methods for preventing the cell death mechanisms that occur post-seizure and repairing this injury after a seizure remain uncertain. Seizures cause serious damage to the hippocampus, and neuronal death is ultimately caused by a series of cell death cascades involving excessive inflammation, glial activation, apoptosis, oxidative stress, and zinc accumulation . Here, wCerebrolysin is neuropeptide extracted from porcine brains and has been used as a nootropic drug . It is kTo evaluate the neuroprotective effects of cerebrolysin, we first injected cerebrolysin once a day for 1 week after a seizure and then confirmed the neuroprotective effects. Then, we performed several histological evaluations after pilocarpine-induced seizure. We next performed staining for NeuN, a specific marker of live neurons, and cleaved caspase-3, a specific marker for apoptotic cells, to confirm the effects of cerebrolysin on neuronal death after pilocarpine-induced seizure. It is already well known that the number of live neurons decreases after a seizure . Also, iIn addition to directly damaging neurons, other cells in the region are also affected by seizure. Epileptic seizure-induced neuroinflammation is triggered by activated glial cells, which include microglia and astroglia . The damPilocarpine-induced seizure triggers glial activation and promotes the production of various inflammatory mediators, thus initiating a cascade of inflammatory processes in the hippocampus . The relNext, we confirmed that the observed reduction in neuronal death was associated with an increase in apoptosis induction through cleaved caspase-3 staining, which detects apoptotic cells. In the seizure-operated groups, the cerebrolysin-administered group showed a significant decrease in the number of apoptotic cells in the hippocampal CA1 and CA3 regions compared to the vehicle group. Cerebrolysin attenuates hippocampal neuronal death and apoptotic cell damage by increasing the concentration of the protease “furin,” which upregulates BDNF levels . IncreasSeveral studies have demonstrated that neurotrophic factors, including NGF and BDNF, are elevated after status epilepticus, which promotes the survival of neurons post-seizure . Thus, wIt is well-established that neuronal death occurs in the brain after a seizure , but thein vivo half-life and uncertain BBB permeability. BDNF is relatively unstable, and only a small fraction can cross the BBB after administration. If the level of administered BDNF is too small due to its short half-life and limited permeability, it may not show observable neurotrophic effects , is a mixture involving fragments of different neurotrophic factors, including BDNF. It has been demonstrated that BBB permeability promotes long-lasting BDNF supply to the brain (Despite BDNF showing potential neuroprotective effects in stroke, traumatic brain injury, and Alzheimer’s disease, the therapeutic delivery of BDNF has many obstacles related to its short  effects . The comhe brain . TherefoThe present study found that the administration of cerebrolysin decreased seizure-induced neuronal death and glial activation by increasing BDNF levels. Although the precise mechanism through which cerebrolysin promotes increased BDNF production and downregulates microglial activation after a seizure remains unclear, the present study suggests that the administration of cerebrolysin can be a useful therapeutic agent to prevent neuronal death in this setting. However, substantial further research is needed to determine the mechanism by which cerebrolysin increases BDNF and promotes other neuroprotective outcomes.All datasets presented in this study are included in the article/The animal study was reviewed and approved by the Committee on Animal Habitation.DK researched the data and reviewed and edited the manuscript. BC reviewed and edited the manuscript. SL, AK, JJ, DH, BK, MP, HS, and HC researched the data. M-SL and SS contributed to the discussion and wrote, reviewed, and edited the manuscript. HS, HC, M-SL, and SS take full responsibility for the manuscript and its originality. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


After, the explants were cultured in M199 for 24 h to finally collect the media for miRNA analyses using GeneChip miRNA 4.0 Array (Affymetrix). Fifteen differentially expressed  < 0.05 and Fold-change ≥ 2) miRNAs (11 down- versus 4 up-regulated) were identified (the most in the media of uterine explants incubated with SP from post-SRF). Bioinformatics analysis identified that predicted target genes of dysregulated miRNAs, mainly miR-34b, miR-205, miR-4776-3p and miR-574-5p, were involved in functions and pathways related to immune response. In conclusion, SP is able to elicit changes in the miRNAs profile secreted by female genital tract, ultimately depending SP-composition.The seminal plasma (SP) modulates the female reproductive immune environment after mating, and microRNAs (miRNAs) could participate in the process. Considering that the boar ejaculate is built by fractions differing in SP-composition, this study evaluated whether exposure of mucosal explants of the sow internal genital tract  to different SP-fractions changed the profile of explant-secreted miRNAs. Mucosal explants retrieved from oestrus sows ( Mounting evidence suggests that seminal plasma (SP), the complex fluid secreted mainly by the male accessory sexual glands, is not a mere sperm carrier, but it also plays a crucial role in other reproductive physiology events ,2,3. In In pigs, in vivo studies have revealed that SP is able to modulate the maternal environment, interacting with the female reproductive tract and modifying the expression of certain genes, mainly those that are immune response-related ,11. WhilThe role of miRNAs as regulators of several biological processes, including reproductive functions, has attracted significant attention in the last years ,17,18,19Against this background, the present study evaluated whether in vitro exposure of mucosal explants of the internal genital tract of oestrus sows  and isthmus) to SP from different fractions , the post-SRF) or a composite (recomposed entire ejaculate (EE)), changed the profile of secreted miRNAs. n = 72) were retrieved from the two side-segments (right and left) of the uterus, UTJ and isthmus (four explants per each side-segment) of three sow (24 explants per sow) and were individually cultured in Medium 199 (M199) at 38 °C in 5% CO2 in air in a humidified incubator  under continuous gentle shaking  for 60 min. Thereafter, the four explants from the same segment/side/sow were separately subjected to the following treatments (as described below): (1) M199 (Control), and (2) M199 supplemented (1:40 v/v) with pooled SP from (2a) the SRF, (2b) the post-SRF or (2c) the recomposed EE. After a 16 h incubation period, the media were harvested and replaced with 1 mL of fresh M199 and incubated under the same conditions for a further 24 h, to finally collect the medium (mixing the medium of the left and right sides from the same segment/treatment/sow (2 mL)), which was frozen in liquid nitrogen and stored at −80 °C  until being examined for miRNA analysis profiling. Mucosal explants .The chemicals used in the experiments were of analytical grade. Unless otherwise stated, all reagents were acquired from Merck KgaA  and the media were prepared under sterile conditions within a laminar flow hood . 6 spermatozoa/mL, 70% of them motile and 75% of them morphologically normal).Ejaculates used in the experiment were collected from five healthy, mature (2–3 years old) and fertile boars of different crossbreeds (Landrace × Large-White) belonging to a center for artificial insemination . One ejaculate per each boar was collected in two separate fractions using the gloved-hand method: the SRF and the post-SRF. A representative proportion of each fraction was thereafter mixed to mimic a recomposed EE. The ejaculates used in the experiment fulfilled the standards of quantity and quality for commercial AI-doses ), in order to obtain the SP. The resultant SP samples were examined microscopically  to ensure they were sperm-free. Thereafter, the SP-samples were pooled per fraction and EE, stored in conical tubes (15 mL) and shipped in insulated containers with dry ice to the Laboratory, where they were stored at −80 °C (Ultra-Low Freezer). Right after manual semen collection, samples from each of the two ejaculate fractions and the recomposed EE were double centrifuged , and sows returned to recovering crates after intramuscular administration of a long-acting amoxicillin suspension (15 mg/kg body weight). Endometrial  and endosalpingeal (UTJ and isthmus segments) explants were obtained from three multiparous (3–6 parity) healthy crossbred (Landrace × Large White) sows from a commercial farm . The sows were checked for signs of oestrus (twice per day), tested by experienced staff by applying manual back-pressure on the standing sows during snout-to-snout contact with a mature boar. Once sows showed a standing oestrus reflex, animals were subjected to surgery in a specific surgical room located on-farm. Sows were sedated by administration of azaperone  and general anesthesia was induced using sodium thiopental . Anesthesia was maintained with 3% to 5% isoflurane . Sows were subjected to mid-ventral laparotomy, and after exposure of the reproductive tract, ovaries were morphologically examined, confirming the presence of pre-ovulatory 5–8 mm follicles. The surgical procedure followed the protocol described by Martinez et al. . Then, s3 . The explants were cultured for 60 min at 38 °C in a humidified atmosphere containing 5% CO2 (CO₂ Incubator C60) with gentle shaking  prior to each treatment, as described in the Experimental Design Section. The media were collected for storage and an aliquot was indirectly analyzed for cell viability of the explant through measurement of lactate dehydrogenase (LDH) activity using an automated analyzer . The LDH activity  was in uterus: 13.52 ± 0.17 mU/mg, in UTJ: 11.45 ± 0.2 mU/mg, and in isthmus: 26.32 ± 0.98 mU/mg. Under sterile conditions, within a laminar flow hood cabinet , explants (30–40 mg of tissue each) were each transferred individually into a well of a 24-well plate  with 1 mL of culture medium per well (M199), supplemented with 0.1% bovine serum albumin, 0.075 g/L penicillin G, 0.05 g/L streptomycin sulphate and 2.2 g/L NaHCOg at 4 °C for 15 min). Two phases were obtained and 400 μL of the upper phase was extracted and mixed with 600 μL of 100% ethanol. Then, the resultant mixture (700 μL) was placed into a 2 mL tube with RNeasy MinElute spin column and centrifuged (8000× g at RT for 15 s) to allow the RNA to attach onto the column membrane, discarding the flow-through. The process was repeated with the rest of the sample with the same column. Thereafter, the column was successively washed with RWT buffer (700 μL), RPE buffer (500 μL) and 80% ethanol (500 μL), centrifuging (8000× g at RT for 15 s (RWT and RPE) or for 2 min (ethanol)) and discarding the flow-through each time. Lastly, the column was centrifuged , removing the flow-through, and the RNA was obtained by adding 14 μL nuclease-free water onto the column membrane and centrifuging . Total RNA concentration and purity were assessed by NanoDrop® 1000 (Thermo Fisher Scientific) and quality was determined with microfluidic electrophoresis . Only samples with high RNA concentration were stored at −80 °C for its further analysis, because miRNA profiling required a minimum concentration of 16.25 ng/μL. RNA integrity number (RIN) values were 2.36 ± 0.08 (mean ± SEM) and the A260/A280 ratio was ˃1.7 in all cases. These low RIN values have been demonstrated to exert negligible or no effect on miRNA profiling analysis due to the robust stability of miRNAs [A commercially available kit specifically designed to isolate small RNAs in fluids  was used to extract RNA from our samples. In brief, 200 μL of each sample was thawed on ice, mixed  with QIAzol Lysis Reagent, incubated (5 min at RT) and then 200 μL of chloroform was added. The resulting sample was mixed by agitation (15 s at RT), incubated (3 min at RT) and centrifuged  were utilized to perform the miRNA expression profiling. An Affymetrix® FlashTagTM Biotin HSR RNA Labelling Kit (Thermo Fisher Scientific) was used to create Biotin-labeled RNA. In brief, isolated RNA (130 ng) were subjected to poly-A tail incorporation to the 3’-end, and then a biotin-labeled 3DNA molecule was linked to the 3′-end by a DNA ligase. The resultant biotin-labeled RNA samples were hybridized to the GeneChip miRNA 4.0 arrays in an Affymetrix® Oven 455 (Thermo Fisher Scientific) (18 h at 48 °C with 60 rpm rotation). After hybridization, miRNA 4.0 arrays were washed, stained and scanned by the Affymetrix® GeneChipTM Hybridization Wash and Stain Kit (Thermo Fisher Scientific), Affymetrix® Fluidic Station 450 (Thermo Fisher Scientific) and Affymetrix® GeneChip Scanner 3000 7G (Thermo Fisher Scientific), respectively. Affymetrix® GeneChip Command Console Software , and further analyzed using the Affymetrix® Transcriptome Analysis Console software , after normalizing the intensity by the robust multi-array average. Three comparisons were performed per explant, as follows: SP from the SRF versus control, SP from post-SRF versus control and SP from recomposed EE versus control. p-values were adjusted through the Benjamini–Hochberg False Discovery Rate (FDR). miRNAs that fulfilled the criteria  ≤ −2.0) were extracted as significantly up- or down-regulated miRNAs. Principal Component Analysis (PCA) and hierarchical clustering were performed to check grouping of samples based on dysregulated miRNAs using Affymetrix® Transcriptome Analysis Console software v4.0. The raw datasets were deposited at Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/) with the identifier (GSE149079).CEL-files of raw data were produced with the Affymetrixhttp://mirdb.org) [http://www.cytoscape.org/) application ClueGO v2.0.3. Target genes of dysregulated miRNAs were predicted using miRDB (rdb.org) . Gene Onp-value < 0.05 (criterion 1). In the following increasingly restrictive criteria, FDR was used and considered to be significant when the q-value threshold was <0.1 (criterion 2) or <0.05 (criterion 3). Three statistical criteria were applied during screening of differentially expressed (up/down) miRNAs in the culture media harvested from the three different explants of the female genital tract examined , following exposure to different treatments  in relation to controls . Regardless of the statistical criteria applied, miRNAs were firstly selected using a FC cut-off of ≤−2 and ≥2. The first criterion consisted of the conventional statistical significance level set at When criterion 1 was used, the hierarchical cluster analysis applied to the miRNA expression profiles of each explant resulted in a clear separation between treatments and controls, showing that the three sows had the same pattern . Three cp-value < 0.05), and a lower amount of differentially expressed miRNAs was identified using criterion 2 (FDR < 0.1: 55 miRNAs), or criterion 3 (FDR < 0.05: 41 miRNAs). Regardless of the statistical criteria used, the number of downregulated miRNAs was higher than the number of miRNAs upregulated in the culture medium from all explants and treatments compared with their respective controls. Similarly, differential expression of most miRNAs was higher in utero- and UTJ- than in isthmus-media  in the medium from each explant and treatment applying the three statistical criteria are shown in us-media .p-value < 0.05) are listed in p-value, FC and whether they were up- or down-regulated). Here, the number of overlapping differentially expressed miRNAs in the medium of each explant is summarized in Venn diagrams ). Applying this statistical cut-off, none of the previous differentially expressed miRNA was identified in the medium from cultured isthmus. A small number of upregulated miRNAs was found in all uterine media and only in the medium of UTJ exposed to SP from SRF  reducing the number of differentially expressed miRNAs  are presented in http://www.mirbase.org). To reduce the number of false positives, we listed only potential target genes with a score ≥0.9 [Among the 15 differentially expressed miRNAs (criterion 3), the predicted target genes for eight of them were found using the miRNA-target database (miRDB). Since the miRNA-target database has been developed for model organisms (mainly humans), the porcine sequences of those miRNAs described were compared to their human homologs, confirming their high homology  to simulate the dilution rate of commercial AI-dose) for a 16 h period, due to the period expected for boar semen to be exposed to the lining of the genital epithelium of the sow, considering the intervals of the two AI performed per oestrus conventionally practiced elsewhere . It is wThe present study aimed to identify the main dysregulated miRNAs in media of uterus, UTJ and isthmus exposed to SP, and a strict filter criterion was applied (FDR < 0.05 and ≥ 2.0-FC ≤ −2.0). The first contribution of the present study was that no differentially expressed miRNAs were identified in isthmus culture medium, depicting that SP could not exert any influence on the female genital tract at that level. Our results would support the hypothesis that, under physiological conditions, SP does not reach the oviduct in pigs ,52. TherFocusing on miRNA transcriptome profiling of uterine and UTJ media, 15 differentially expressed miRNAs (excluding repeated miRNAs) were identified. The results indicated at first sight that the SP mainly contributed to a global downregulation of miRNA expression in the uterus and UTJ. It is worth noting that, in contrast to the general assumption that miRNAs repress gene expression in varying degrees , a transCXCL5, IL1RAP and IL5RA were found among the genes targeted by three dysregulated miRNAs (miR-4776-3p (down), miR-34b (down) and miR-574-5p (up), respectively) identified in uterine-media. Collectively, these results could contribute to explaining how SP-miRNAs underlie the regulation of cytokine-related gene expression in the female genital tract.One of the main findings of the current study was that the dysregulated miRNAs identified in UTJ and utero-harvested media differed depending on the SP-source  to which explants were exposed. In the pig, as in the human  and the This study identified, for the first time, several dysregulated miRNAs in uterus and UTJ-culture media obtained during in vitro culture for 24 h after different SP-treatments, whose specific function, biological relevance and/or predicted target genes have not yet been reported. Specifically, regardless of the SP-source used, miR-4601, miR-2313-3p and miR6460 were downregulated in UTJ- and utero-media, and miR-4384 was downregulated in utero-media. Similarly, our results demonstrated that miR-3944-3p was downregulated in the culture medium of UTJ incubated with SP from EE. Finally, miR-1713 and miR-3104-5p were upregulated in the media of uterus incubated with SP from EE, and in that of the UTJ incubated with SP from SRF, respectively. Considering that SP modulates the female reproductive immune environment, conditioning the success of fertilization, embryo implantation and pregnancy [IL1RAP, one of the miR-34b target-genes, are higher in the endometrium of pregnant than of non-pregnant sows [ALOX15, TRIM14, PAK5 and USP25, were identified among its potential target-genes, being related to the immune response, as indicated by DAVID and KEGG databases. This study also identified dysregulated miRNAs in uterus and UTJ-media whose biological relevance and predicted target genes were previously reported. miR-34b and miR-4776-3p were downregulated in utero-media, regardless of the SP-source to which the uterus was exposed. Studies performed in humans have reported that dysregulation of miR-34b expression is related to endometrial cancer and endometriosis ,62,63, aant sows . The resThe miR-92b-5p was upregulated in the culture media of uterus’ exposed to SP from SRF and post-SRF. Salilew-Wondim et al.  revealedCOL3A1, MRC1, PIK3CG, SIRT1, NAFTC3 and PLCB1, were related to the immune response. Since a close link between the immune response and cancer has also been widely described [Our results also showed that miR-205 and miR-205a-5p were downregulated in the media of uterus’ exposed to SP from post-SRF and EE. The relevance of miR-205 on female genital tissues has been extensively reported in humans, since a dysregulation of this miRNA has been associated with endometrial cancer ,74,75. Described ,66, it iFinally, the results also evidenced that miR-23a-5p and miR-574-5p were respectively, down- and up-regulated in the culture media of UTJ and uterus’ incubated with SP from post-SRF. Previously, reports have shown that both miRNAs could be involved in immune functions. Specifically, it was revealed that miR-23a is involved in regulating cytokine production in macrophages , and in In conclusion, based on the information described above, our study is the first one to demonstrate that SP is able to elicit changes in the expression profile of miRNAs in UTJ and utero, ultimately depending on the SP-composition. Since it has been reported that SP modulates female reproductive tract immune response at the peri-conception period, this study provides new insights into the identification of known and novel miRNAs that could be responsible for modulating that process. This preliminary study could serve as a basis for further studies in order to contribute to the understanding of the role played by these miRNAs in reproductive processes. Furthermore, these results could be valuable for AI centers to improve the efficiency of AI semen dose, since they provide a new vision of the role played by the SP of the different ejaculate fractions on the reproductive tissues of the sows.